Method for purifying antibodies

ABSTRACT

Disclosed here includes a method for purifying a biologic composition, comprising diafiltering the biologic composition into a composition comprising phosphate buffered saline (PBS) to obtain a purified composition. The method disclosed here can be particularly useful for removing one or more impurities from the biologic composition, such as bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (Bis-tris).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of PCT/US2015/042241, filed Jul. 27,2015, which claims the benefit of U.S. Provisional Patent ApplicationNo. 62/028,994 filed Jul. 25, 2014, the content of which is incorporatedherein by reference in its entirety.

BACKGROUND OF THE INVENTION

Known processes for purifying monoclonal antibodies and other biologicalmaterials are often required to remove unwanted impurities, which isparticularly important when the biologic is produced for therapeuticuses. One way to remove impurities is through diafiltration.Diafiltration is known in the art and described in, for example, WayneP. Olson, Separations Technology: Pharmaceutical and BiotechnologyApplications (Interpharm Press 1995); Munir Cheryan, Ultrafiltration andMicrofiltration Handbook (2d ed. CRC Press 1998); Stefan Behme,Manufacturing of Pharmaceutical Proteins (Wiley-VCH 2009); and Glyn N.Stacey, Medicines from Animal Cell Culture (John Wiley 2007), all ofwhich are incorporated herein by reference in their entireties.

Ch14.18 (also referred to herein as “dinutuximab”) is an anti-GD₂monoclonal antibody and has been described in Gillies et al., Journal ofImmunological Methods 125:191-202 (1989), which is incorporated hereinby reference in its entirety. When using the ch14.18 antibody fortherapeutic purposes, it is important to remove impurities such asbis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (Bis-tris) to ensurethe safety and effectiveness of the monoclonal antibody. Thus, there isa need for methods of removing unwanted impurities from biologiccompositions.

SUMMARY OF THE INVENTION

Many embodiments of the invention described herein relates to a methodfor purifying a biologic composition, comprising diafiltering thebiologic composition with phosphate buffered saline (PBS) to obtain apurified composition.

In one embodiment, the biologic composition comprises at least oneisolated protein.

In one embodiment, the isolated protein is a monoclonal antibody. In oneembodiment, the monoclonal antibody is ch14.18.

In one embodiment, the biologic composition further comprises at leastone impurity. In one embodiment, the impurity isbis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (Bis-tris). In oneembodiment, the concentration of the Bis-tris in the biologiccomposition is 10 to 50 mM Bis-tris at a pH of 6.3 to 6.7.

In one embodiment, the diafiltering removes at least 50% of the Bis-trisfrom the biologic composition. In one embodiment, the diafilteringremoves at least 70% of the Bis-tris from the biologic composition.

In one embodiment, the concentration of the PBS is 10 to 50 mM SodiumPhosphate and 100 to 200 mM NaCl.

In one embodiment, the monoclonal antibody is concentrated to aconcentration of at least 2.0 to 5.0 AU before being diafiltered intothe composition comprising PBS. In one embodiment, the monoclonalantibody is concentrated to a concentration of at least 4.0 to 6.0 AUbefore being diafiltered into the composition comprising PBS.

In one embodiment, the method further comprises isolating and purifyingthe monoclonal antibody using at least one chromatography column.

In one embodiment, the method comprises isolating and purifying themonoclonal antibody using at least one affinity chromatography column,at least one cation exchange chromatography column, and/or at least oneanion exchange chromatography column. In one embodiment, the anionexchange chromatography column is a Capto™ adhere column.

In one embodiment, the monoclonal antibody is eluted from the Capto™adhere column using a composition comprising Bis-tris.

In one embodiment, at least three volume units of the biologiccomposition is diafiltered into one volume unit of the compositioncomprising PBS. In one embodiment, at least five volume units of thebiologic composition is diafiltered into one volume unit of thecomposition comprising PBS.

In one embodiment, the purified composition is further diafiltered intoa composition comprising histidine.

Further described is a method for purifying a monoclonal antibody,comprising:

-   -   (a) passing a first composition comprising the monoclonal        antibody through an affinity chromatography column to obtain a        second composition comprising the monoclonal antibody; (b)        lowering the pH of the second composition to obtain a third        composition; (c) washing the third composition with a        solvent-detergent to obtained a fourth composition; (d) washing        the fourth composition to remove the solvent-detergent to obtain        a fifth composition; (e) passing the fifth composition through a        cation exchange chromatography column to obtain a sixth        composition comprising the monoclonal antibody; (f) subjecting        the sixth composition to nano-filtration to obtain a seventh        composition; (g) passing the seventh composition through an        anion exchange chromatography column to obtain an eighth        composition comprising the monoclonal antibody and Bis-tris;        and (h) diafiltering the eighth composition into a composition        comprising PBS to obtain a ninth composition comprising the        monoclonal antibody but substantially free from Bis-tris.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows weak cation exchange HPLC Analysis of ch14.18 afterdiafiltration with PBS. Line A represents ch14.18 diafiltered with PBS.Line B represents ch14.18 diafiltered into HBS, both monitored at 215nm.

FIG. 2 shows sample of 25 mM Bis-tris loaded onto the weak cationexchange HPLC column, and demonstrates that the peak with a retentiontime of ˜7 minutes is due to Bis-tris. Evaluation of 25 mM Bis-tris byweak cation exchange. Line A indicates the absorbance monitored at 215nm. Line B indicates the absorbance monitored at 280 nm.

FIG. 3 shows weak cation HPLC of Capto™ adhere eluate pool from new orused resin, and demonstrates that the peak at ˜7 minutes is due toBis-tris and not to some contaminant on the column from prior usage.Samples of Capto™ adhere eluate pools, with no ch14.18 derived from newor used chromatography columns. Line A is for sample from the newcolumn. Line B is from the used column.

FIG. 4 shows weak cation exchange HPLC chromatography of ch14.18, spikedwith contaminants prior to diafiltration into PBS. Line A representsch14.18 with no contaminant spike. Line B represents ch14.18 spiked withtributyl phosphate and polysorbate 80. Line C represents ch14.18 spikedwith methotrexate. None of the trace additives from the purificationprocess are responsible for the peak at ˜7 minutes (FIGS. 1 & 2).

FIG. 5 provides a schematic depicting one embodiment of a process flowchart for purifying a monoclonal antibody (e.g., ch14.18). Thispurification process was initially developed by the National CancerInstitute (NCI) (see Example 2) and subsequently improved by UnitedTherapeutics Corp (see Example 1).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Many embodiments described herein relate to a method for purifying abiologic composition, comprising diafiltering the biologic compositionwith phosphate buffered saline (PBS), to obtain a purified composition.

Biologic Composition

Many biologic compositions known in the art can be purified using themethods described herein. The biologic composition can comprise at leastone material created biologically rather than chemically synthesized,including proteins, nucleic acids, cells, tissues, vaccines, and bloodor a component thereof.

The biologic composition can comprise, for example, at least oneisolated protein including a recombinant protein. The biologiccomposition can comprise, for example, at least one isolated nucleicacid. The biologic composition can comprise, for example, at least onemonoclonal antibody. The biologic composition can comprise, for example,at least one chimeric, altered, or humanized antibody.

In one particular embodiment, the biologic composition comprises atleast one ch14.18 monoclonal antibody, although other antibodies andbiologics can be purified by the teaching disclosed herein.

The biologic composition can comprise, for example, at least oneimpurity. The impurity can be, for example, an undesirable salt such asBis-tris. The concentration of the Bis-tris impurity in the biologiccomposition can be, for example 1 to 100 mM Bis-tris, or 10 to 50 mMBis-tris, or 20 to 40 mM Bis-tris. The pH can be, for example, 6 to 7,or 6.3 to 6.7, or 6.4 to 6.6.

Diafiltration with PBS

In some embodiments, the method described herein comprises diafilteringat least two, at least three, at least four, at least five, or at leastsix volume units of the biologic composition into one volume unit ofPBS.

In some embodiments, the biologic composition comprises undesirableBis-tris, and the method described herein comprises removing at least30%, at least 50%, at least 70%, at least 90%, or at least 95% of theBis-tris from the biologic composition by means of PBS diafiltration.

The concentration of the PBS can be, for example, 10 to 50 mM SodiumPhosphate, and 100 to 200 mM NaCl.

In some embodiments, prior to being diafiltered with PBS, the biologiccomposition is concentrated first.

In some embodiments, after the PBS diafiltration, the biologicalcomposition is diafiltered into a formulation comprising 20 mMhistidine, 150 mM NaCl, and 0.05% Tween 20 saline (HBS).

Isolation of Monoclonal Antibody

In several embodiments described herein, the method also comprisesisolating and purifying the monoclonal antibody before the PBSdiafiltration. The monoclonal antibody can be isolated and purified by,for example, at least one chromatography column. The monoclonal antibodycan be isolated and purified by, for example, at least one affinitychromatography column, at least one cation exchange chromatographycolumn, and/or at least one anion exchange chromatography column. Theanion exchange chromatography column can be, for example, a Capto™adhere column.

Capto™ adhere column is known in the art and a commercially availablefrom GE Healthcare Life Sciences. It is a multimodal medium forintermediate purification and polishing of monoclonal antibodies aftercapture on Protein A medium by packed bed chromatography. Someembodiments described herein comprises purifying a monoclonal antibodyusing Capto™ adhere column, wherein the monoclonal antibody is elutedwith a composition comprising Bis-tris buffer. The eluted antibodyformulation must then be diafiltered into a formulation comprising PBS,as discussed above, and optionally further diafiltered into aformulation comprising HBS.

The methods described herein can further comprise, for example,concentrating the biologic composition comprising harvested monoclonalantibody.

The methods described herein can further comprise, for example,diafiltering the biologic composition into Protein A equilibrationbuffer. In a particular aspect, the Protein A equilibration buffercomprises 25 to 100 mM Sodium Phosphate, 0.5 to 2.0 M NaCl, with pH 7.0to 8.0.

The methods described herein can further comprise, for example, passingthe biologic composition through an affinity chromatography column, suchas a Protein A affinity column.

The methods described herein can further comprise, for example, viralinactivation by lowering the PH of the biologic composition.

The methods described herein can further comprise, for example, viralinactivation by washing the biologic composition with asolvent-detergent. In a particular aspect, the solvent-detergentcomprises 10 to 25% Polysorbate (TWEEN), 5 to 10% Tributyl Phosphate.

The methods described herein can further comprise, for example, washingthe biologic composition to remove any solvent-detergent.

The methods described herein can further comprise, for example,diafiltering the biologic composition into 50 HS equilibration buffer.In a particular aspect, the 50 HS equilibration buffer comprises 5 to 20mM Citrate, 5 to 30 mM Phosphate, 15 to 100 mM Sodium Chloride, with pH4.0 to 5.5.

The methods described herein can further comprise, for example, passingthe biologic composition through a cation exchange affinitychromatography column such as a 50 HS column.

The methods described herein can further comprise, for example,subjecting the biologic composition to nano-filtration.

The methods described herein can further comprise, for example, passingthe biologic composition through an anion exchange affinitychromatography column, such as a Capto™ adhere column before the PBSdiafiltration. The monoclonal antibody can be eluted from the Capto™adhere column using a Bis-tris buffer.

The methods described herein can further comprise, for example,diafiltering the biologic composition into a histidine buffer after thePBS diafiltration.

In one embodiment, the methods described herein comprises the followingsteps:

-   -   (a) passing a first composition comprising the monoclonal        antibody through an affinity chromatography column to obtain a        second composition comprising the monoclonal antibody;    -   (b) lowering the pH of the second composition to obtain a third        composition;    -   (c) washing the third composition with a solvent-detergent to        obtained a fourth composition;    -   (d) washing the fourth composition to remove the        solvent-detergent to obtain a fifth composition;    -   (e) passing the fifth composition through a cation exchange        chromatography column to obtain a sixth composition comprising        the monoclonal antibody;    -   (f) subjecting the sixth composition to nano-filtration to        obtain a seventh composition;    -   (g) passing the seventh composition through an anion exchange        chromatography column to obtain an eighth composition comprising        the monoclonal antibody and Bis-tris; and    -   (h) diafiltering the eighth composition into a composition        comprising PBS to obtain a ninth composition comprising the        monoclonal antibody but substantially free from Bis-tris.

WORKING EXAMPLES Example 1—Development of Weak Cation Exchange HPLCAssay

In the development of the ch14.18 purification process, an objective wasto improve the purification process which was initially developed by theNational Cancer Institute (NCI) (see FIG. 5 for the process initiallydeveloped by the NCI). A further objective was to upgrade tochromatography resins which had better capacity, better flow properties,or were tolerant of harsher chemicals that could be useful to sanitizethe resins prior to the next use (e.g., Capto™ adhere resin from GEHealthcare).

For the last two chromatography steps (see Sections 18 and 19 of FIG.5), NCI used a Superdex 200 size-exclusion chromatography column toremove aggregates and dimers of ch14.18, followed by a Q Sepharose anionexchange resin as a polishing resin. They were upgraded to a resin thatremoves viruses, aggregates, and traces contaminants—the Capto™ adhereresin from GE Healthcare (see e.g., GE Healthcare Life Sciences,Instructions 28-9064-05 Capto™ adhere Affinity Chromatography productmanual, which is incorporated herein by reference in its entirety and isavailable athttps://www.gelifesciences.com/gehels_images/GELS/Related%20Content/Files/1334667780708/litdoc28906405_20120420104439.pdf).This resin is a mixed mode resin, a combination of anion exchange andhydrophobic interaction. It was operated in the flow through mode, inwhich the contaminants bind to the resin, while the ch14.18 monoclonalantibody did not bind and remained in the flow-through.

At the same time that the purification of ch14.18 was being revised,quality indicating assays were also being developed. One of these was aweak cation exchange chromatography HPLC assay. For this, the TSKGELCM-3SW column from TOSOH was used to assess the purity of ch14.18. Forthis analysis, the antibody bound to the column and eluted the column asa salt gradient was run through the column. The column was equilibratedin a low salt sodium phosphate buffer (buffer A) and the gradient wasgenerated with an increased concentration of sodium phosphate/sodiumperchlorate buffer (Buffer B). With this separation method the columneffluent was monitored at 215 nm and the ch14.18 peak eluted from thecolumn at approximately 15-19 minutes after injection. The retentiontime for ch14.18 varied as the method and gradient were being optimized.During the development of the assay, various samples of ch14.18 weretested and in some of them a fairly large peak, having a retention timeof ˜7.5 minutes was observed. In trying to identify this contaminant,samples of ch14.18 were spiked with contaminants that are added early inthe manufacturing process (see FIG. 1). A sample of ch14.18 which didnot contain the contaminant was spiked with methotrexate or tributylphosphate and polysorbate 80. None of these contaminants appeared to bethe source of the peak with the retention time of 7.5 minutes.

Through tracing the source of the samples and which step in thepurification process the sample was derived from, it was noted that thepeak at issue was only seen after Capto™ adhere chromatography, whichwas the final polishing step in the purification process. It wassuspected that the contaminant was some trace contaminant, such as hostcell protein from the cell culture process that was co-purifying andconcentrating on the column. Samples from mock runs on a Capto™ adherecolumn which had been loaded on a used column or a new column wereevaluated by weak cation exchange chromatography HPLC (see FIG. 2). Thecontaminant peak at 7.5 minutes was found in both samples atapproximately the same concentration, indicating that the contaminantwas not derived from upstream contaminants.

By tracking the origin of the samples of ch14.18 which contained thesamples and those that did not, it became apparent that the contaminantwas Bis-tris which is the buffer salt used for the Capto™ adherepurification step (see FIG. 3).

Having identified the contaminant, investigated next was the reason thatsome of the purified ch14.18 samples, which had been through the entirepurification process, contained the Bis-tris peak, whereas others didnot. Also being developed is a final antibody formulation in 20 mMhistidine, 150 mM NaCl, 0.05% Tween 20 saline (HBS), which wastransitioned from a formulation in phosphate buffered saline (PBS) usedby NCI. It was noted that if the Capto™ adhere product pool containingch14.18 was diafiltered directly into the HBS buffer, the Bis-tris peakdid not diafilter out of the retentate pool. However, if this pool wasfirst diafiltered into phosphate buffered saline and then into the HBSbuffer, the Bis-tris was completely removed from the solution. FIG. 4shows ch14.18 from the Capto™ adhere chromatography step that had beendiafiltered into HBS or diafiltered with PBS

In conclusion, the Capto™ adhere column is commonly used in thebiotechnology industry for the purification of monoclonal antibodies. Ithas utility for the removal of trace contaminants such as residualProtein A ligand, residual host cell DNA, residual host cell proteins,antibody aggregates, and viruses. Diafiltration with PBS was proved tobe an effective way to remove the Bis-tris impurities prior to the finalformulation of a monoclonal antibody such as ch14.18.

Example 2—Process Flow Chart

A Process Flow Chart is provided in FIG. 5 which depicts one embodimentfor purifying a monoclonal antibody (e.g., Ch14.18). The processdepicted is not meant to be strictly limited and may be adjusted inaccordance with the particular needs and circumstances of the process athand. The process of FIG. 5 generally refers to the following steps:

Section 8. Initial pooling and filtration of harvest of the monoclonalantibody (e.g., crude harvest).

Section 9. Passing the harvests of Section 8 through an affinitychromatography column (e.g., a Protein A chromatography column) toobtain a second composition comprising the monoclonal antibody.

Section 10. Inactivating viruses by lowering the pH of the secondcomposition to obtain a third composition. The third composition is thenwashed with a solvent-detergent to obtained a fourth composition, whichis then washed again to remove the solvent-detergent to obtain a fifthcomposition.

Section 11. Passing the fifth composition through a cation exchangechromatography column (e.g., SP Sepharose FF Chromatography) to obtain asixth composition comprising the monoclonal antibody. The Protein Achromatography can be repeated (Section 12), followed by a repeating ofthe viral inactivation (Section 13), followed by a repeating of thecation exchange chromatography (e.g., Sepharose) (Section 14). TheSepharose runs are then pooled to form the sixth composition.

Section 16. Subjecting the sixth composition to nano-filtration tofurther remove viruses to obtain a seventh composition.

Sections 17, 18, 19. Concentrating the monoclonal antibody by passingthe seventh composition through a Superdex 200 size-exclusionchromatography column to remove aggregates and dimers of the monoclonalantibody. The composition is then passed through a Q Sepharose anionexchange resin to obtain an eighth composition comprising the monoclonalantibody.

Section 20. Concentration and Final Filtration. The eighth compositionis then subjected to concentration and final filtration, includingdiafiltration into a composition comprising PBS, to obtain a ninthcomposition comprising the monoclonal antibody.

Additional Embodiments Embodiment 1

A method for purifying a biologic composition, comprising diafilteringthe biologic composition with phosphate buffered saline (PBS) to obtaina purified composition.

Embodiment 2

The method of embodiment 1, wherein the biologic composition comprisesat least one isolated protein.

Embodiment 3

The method of any of embodiments 1 to 2, wherein the biologiccomposition comprises at least one isolated monoclonal antibody such asch14.18.

Embodiment 4

The method of any of embodiments 1 to 3, wherein the biologiccomposition further comprises at least one impurity such asbis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (Bis-tris).

Embodiment 5

The method of any of embodiments 1 to 4, wherein the biologiccomposition further comprises Bis-tris at a concentration of 10 to 50 mMBis-tris at a pH of 6.3 to 6.7.

Embodiment 6

The method of any of embodiments 1 to 5, wherein the diafilteringremoves at least 50%, at least 70%, or at least 90% of the Bis-tris fromthe biologic composition.

Embodiment 7

The method of any of embodiments 1 to 6, wherein the concentration ofthe PBS is 10 to 50 mM Sodium Phosphate and 100 to 200 mM NaCl.

Embodiment 8

The method of any of embodiments 1 to 7, wherein the monoclonal antibodyis concentrated to a concentration of at least 2.0 to 5.0 AU, beforebeing diafiltered into the composition comprising PBS.

Embodiment 9

The method of any of embodiments 1 to 8, comprising isolating andpurifying the monoclonal antibody using at least one chromatographycolumn.

Embodiment 10

The method of any of embodiments 1 to 9, comprising isolating andpurifying the monoclonal antibody using at least one affinitychromatography column, at least one cation exchange chromatographycolumn, and/or at least one anion exchange chromatography column such asCapto™ adhere column.

Embodiment 11

The method of any of embodiments 1 to 10, wherein the monoclonalantibody is eluted from the Capto™ adhere column using a compositioncomprising Bis-tris before being diafiltered with PBS.

Embodiment 12

The method of any of embodiments 1 to 11, wherein at least three volumeunits, least four volume units, least five volume units, or least sixvolume units of the biologic composition is diafiltered into one volumeunit of the composition comprising PBS.

Embodiment 13

The method of any of embodiments 1 to 12, wherein the purifiedcomposition is further diafiltered into a composition comprisinghistidine.

Embodiment 14

A method for purifying a monoclonal antibody, comprising: (a) passing afirst composition comprising the monoclonal antibody through an affinitychromatography column to obtain a second composition comprising themonoclonal antibody; (b) lowering the pH of the second composition toobtain a third composition; (c) washing the third composition with asolvent-detergent to obtained a fourth composition; (d) washing thefourth composition to remove the solvent-detergent to obtain a fifthcomposition; (e) passing the fifth composition through a cation exchangechromatography column to obtain a sixth composition comprising themonoclonal antibody; (f) subjecting the sixth composition tonano-filtration to obtain a seventh composition; (g) passing the seventhcomposition through an anion exchange chromatography column to obtain aneighth composition comprising the monoclonal antibody and Bis-tris; and(h) diafiltering the eighth composition into a composition comprisingPBS to obtain a ninth composition comprising the monoclonal antibody butsubstantially free from Bis-tris.

What is claimed is:
 1. A method for purifying a biologic composition,wherein the biologic composition comprises at least one impurity,wherein the impurity is bis (2-hydroxyethyl)amino-tris(hydroxymethyl)methane (Bis-tris), wherein the method comprisesdiafiltering the biologic composition with phosphate buffered saline(PBS) to obtain a purified composition, wherein the biologic compositioncomprises a ch14.18 monoclonal antibody.
 2. The method of claim 1,wherein the ch14.18 monoclonal antibody is an isolated protein.
 3. Themethod of claim 1, wherein the concentration of the Bis-tris in thebiologic composition is 10 to 50 mM Bis-tris at a pH of 6.3 to 6.7. 4.The method of claim 1, wherein the diafiltering removes at least 50% ofthe Bis-tris from the biologic composition.
 5. The method of claim 1,wherein the diafiltering removes at least 70% of the Bis-tris from thebiologic composition.
 6. The method of claim 1, wherein theconcentration of the PBS is 10 to 50 mM Sodium Phosphate and 100 to 200mM NaCl.
 7. The method of claim 1, wherein the ch14.18 antibody isconcentrated to a concentration of at least 2.0 to 5.0 AU before beingdiafiltered into the composition comprising PBS.
 8. The method of claim1, wherein the ch14.18 antibody is concentrated to a concentration of atleast 4.0 to 6.0 AU before being diafiltered into the compositioncomprising PBS.
 9. The method of claim 1, further comprising isolatingand purifying the ch14.18 antibody using at least one chromatographycolumn.
 10. The method of claim 9, comprising isolating and purifyingthe ch14.18 antibody using at least one affinity chromatography column,at least one cation exchange chromatography column, and/or at least oneanion exchange chromatography column.
 11. The method of claim 10,wherein the anion exchange chromatography column is a multimodal mediumcolumn.
 12. The method of claim 11, wherein the ch14.18 antibody iseluted from the multimodal medium column using a composition comprisingBis-tris.
 13. The method of claim 1, wherein at least three volume unitsof the biologic composition is diafiltered into one volume unit of thecomposition comprising PBS.
 14. The method of claim 1, wherein at leastfive volume units of the biologic composition is diafiltered into onevolume unit of the composition comprising PBS.
 15. The method of claim1, wherein the purified composition is further diafiltered into acomposition comprising histidine.
 16. A method for purifying amonoclonal antibody, comprising: (a) passing a first compositioncomprising the monoclonal antibody through an affinity chromatographycolumn to obtain a second composition comprising the monoclonalantibody; (b) lowering the pH of the second composition to obtain athird composition; (c) washing the third composition with asolvent-detergent to obtain a fourth composition; (d) washing the fourthcomposition to remove the solvent-detergent to obtain a fifthcomposition; (e) passing the fifth composition through a cation exchangechromatography column to obtain a sixth composition comprising themonoclonal antibody; (f) subjecting the sixth composition tonano-filtration to obtain a seventh composition; (g) passing the seventhcomposition through an anion exchange chromatography column to obtain aneighth composition comprising the monoclonal antibody and Bis-tris; and(h) diafiltering the eighth composition into a composition comprisingPBS to obtain a ninth composition comprising the monoclonal antibody butsubstantially free from Bis-tris.
 17. A method for removingbis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (bis-tris) from abiologic composition, comprising diafiltering the biologic compositionwith phosphate buffered saline (PBS) to remove at least 50% of thebis-tris from the biologic composition, wherein the biologic compositioncomprises a ch14.18 monoclonal antibody.